Thermodynamic Analysis of Weak Protein Interactions Using Sedimentation Equilibrium

Yuri V. Sergeev1, Monika B. Dolinska1, Paul T. Wingfield2

1 National Eye Institute, National Institutes of Health, Bethesda, Maryland, 2 National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.13
DOI:  10.1002/0471140864.ps2013s77
Online Posting Date:  August, 2014
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Abstract

Proteins self‐associate to form dimers and tetramers. Purified proteins are used to study the thermodynamics of protein interactions using the analytical ultracentrifuge. In this approach, monomer‐dimer equilibrium constants are directly measured at various temperatures. Data analysis is used to derive thermodynamic parameters, such as Gibbs free energy, enthalpy, and entropy, which can predict which major forces are involved in protein association. Curr. Protoc. Protein Sci. 77:20.13.1‐20.13.15. © 2014 by John Wiley & Sons, Inc.

Keywords: sedimentation equilibrium; weak protein interaction; thermodynamics

     
 
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Table of Contents

  • Introduction
  • Thermodynamics of Protein Association: Theory and Data Analysis
  • Basic Protocol 1: Temperature‐Dependent Sedimentation Equilibrium to Monitor Protein Associate Formation and Determine Dissociation Constants
  • Support Protocol 1: Derivation of Thermodynamic Parameter from Dissociation Constants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Temperature‐Dependent Sedimentation Equilibrium to Monitor Protein Associate Formation and Determine Dissociation Constants

  Materials
  • Protein, purified and of known concentration (0.2 µM)
  • Buffer B (see recipe)
  • Buffer A (see recipe)
  • Reductant TCEP (Thermo Scientific)
  • Dialysis membranes Snake or Slide‐A‐Lyzer dialysis kits (Thermo Scientific)
  • Gel filtration column
  • UV‐Vis spectrophotometer (suppliers include Agilent and Beckman)
  • Centrifugation was carried out using a Beckman Coulter Optima XL‐I analytical ultracentrifuge; absorption optics, an An‐60 Ti rotor, and standard double‐sector centerpiece cells are used (Beckman)
  • Beckman XL‐A/ XL‐I data analysis software V6.04
  • Additional reagents and equipment for dialysis ( appendix 3B) or buffer exchange using a desalting column (unit 8.3) and SDS‐PAGE (see the protocol 1Basic Protocol in unit 10.1)
NOTE: The amount of protein used for sedimentation equilibrium depends mainly on the availability of the protein. Typically, 3 to 5 mg is required.NOTE: Always make sure that the solution has reached the required temperature before starting the measurement. Extra time must be allowed for temperature equilibration if the protein solution has been on ice before the experiment.
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Figures

Videos

Literature Cited

Literature Cited
  Bax, B., Lapatto, R., Nalini, V., Driessen, H., Lindley, P.F., Mahadevan, D., Blundell, T.L., and Slingsby, C. 1990. X‐ray analysis of beta B2‐crystallin and evolution of oligomeric lens proteins. Nature 347:776‐780.
  Bloemendal, H. and de Jong, W.W. 1991. Lens proteins and their genes. Prog. Nucleic Acid. Res. Mol. Biol. 41:259‐281.
  Chan, M.P., Dolinska, M., Sergeev, Y.V., Wingfield, P.T., and Hejtmancik, J.F. 2008. Association properties of betaB1‐ and betaA3‐crystallins: Ability to form heterotetramers. Biochemistry 47:11062‐11069.
  Dolinska, M.B., Sergeev, Y.V., Chan, M.P., Palmer, I., and Wingfield, P.T. 2009. N‐terminal extension of beta B1‐crystallin: Identification of a critical region that modulates protein interaction with beta A3‐crystallin. Biochemistry 48:9684‐9695.
  Dolinska, M.B., Wingfield, P.T., and Sergeev, Y.V. 2012. betaB1‐crystallin: Thermodynamic profiles of molecular interactions. PLoS One 7:e29227.
  Gill, S.C. and von Hippel, P.H. 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319‐326.
  Iyer, K.S. and Klee, W.A. 1973. Direct spectrophotometric measurement of the rate of reduction of disulfide bonds. The reactivity of the disulfide bonds of bovine ‐lactalbumin. J. Biol. Chem. 248:707‐710.
  Lampi, K.J., Oxford, J.T., Bachinger, H.P., Shearer, T.R., David, L.L., and Kapfer, D.M. 2001. Deamidation of human beta B1 alters the elongated structure of the dimer. Exp. Eye Res. 72:279‐288.
  Laue, T.M., Shah, B.D., Ridgeway, T.M., and Pelletier, S.L. 1992. Computer‐aided interpretation of analytical sedimentation data for proteins. 90‐125.
  Lubsen, N.H., Aarts, H.J., and Schoenmakers, J.G. 1988. The evolution of lenticular proteins: The beta‐ and gamma‐crystallin super gene family. Prog. Biophys. Mol. Biol. 51:47‐76.
  McRorie, D.K. and Voelker, P.J. 1993. Self‐Associating Systems in the Analytical Ultracentrifuge. Beckman Instruments, Fullerton, Calif.
  Ross, P.D. and Subramanian, S. 1981. Thermodynamics of protein association reactions: Forces contributing to stability. Biochemistry 20:3096‐3102.
  Sergeev, Y.V. and Hejtmancik, J.F. 1997. A method for determining domain binding sites in proteins with swapped domains: Implications for betaA3‐ and betaB2‐crystallins. VIII:817‐826.
  Sergeev, Y.V., Chirgadze, Y.N., Mylvaganam, S.E., Driessen, H., Slingsby, C., and Blundell, T.L. 1988. Surface interactions of gamma‐crystallins in the crystal medium in relation to their association in the eye lens. Proteins 4:137‐147.
  Sergeev, Y.V., Wingfield, P.T., and Hejtmancik, J.F. 2000. Monomer‐dimer equilibrium of normal and modified beta A3‐crystallins: Experimental determination and molecular modeling. Biochemistry 39:15799‐15806.
  Sergeev, Y.V., Hejtmancik, J.F., and Wingfield, P.T. 2004. Energetics of domain‐domain interactions and entropy driven association of beta‐crystallins. Biochemistry 43:415‐424.
  Slingsby, C. and Bateman, O.A. 1990. Qarternary interactions in eye lens beta‐crystallins: Basic and acidic subunits of beta‐crystallins favor heterologous association. Biochemistry 29:6592‐6599.
  Takata, T., Woodbury, L.G., and Lampi, K.J. 2009. Deamidation alters interactions of beta‐crystallins in hetero‐oligomers. Mol. Vis. 15:241‐249.
  Willoughby, C.E., Shafiq, A., Ferrini, W., Chan, L.L., Billingsley, G., Priston, M., Mok, C., Chandna, A., Kaye, S., and Heon, E. 2005. CRYBB1 mutation associated with congenital cataract and microcornea. Mol. Vis. 11:587‐593.
  Wistow, G., Turnell, B., Summers, L., Slingsby, C., Moss, D., Miller, L., Lindley, P., and Blundell, T. 1983. X‐ray analysis of the eye lens protein gamma‐II crystallin at 1.9 A resolution. J. Mol. Biol. 170:175‐202.
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